The enteroendocrine cells, which comprise approximately 1% of epithelial cells in the gastrointestinal tract, represent the largest population of hormone-producing cells in the body. The enteroendocrine cells share a common lineage with other non-endocrine cell lineages and originate from primitive intestinal stem cells in the intestinal crypts. There are two complementary populations of stem cells, a self-renewing stem cell population and a quiescent stem cell population. The self-renewing stem cells highly express Lgr5 and reside between Paneth cells at the crypt base where Wnt signaling is active whereas quiescent stem cells express Bmi1 and reside at the position 4. Thus the lineage-specific precursor cells are thought to differentiate immediately from the self-renewing Lgr5+ cells. Endocrine precursor cells differentiate toward mature hormone-producing endocrine cells that are classified into at least 15 different terminally differentiated lineages (or subsets) by their expression of specific peptide hormones. Although the molecular mechanisms that regulate the differentiation from the endocrine precursor cells have not been fully characterized, key transcription factors have been implicated in enteroendocrine cell differentiation (Pax4, Pax6, BETA2/NeuorD, Pdx1, Gfi, Nkx2.2 and Sox9). There is a relationship between spatial orientation of the enteroendocrine cells and their differentiation process. Previous reports on characterization of entroendocrine cell differentiation using BrdU incorporation, morphological and immunohistochemical methods demonstrated that the majority of enteroendocrine cells complete the differentiation process within the crypt and migrate upward along the villus as mature hormone-producing cells. However, a small population of enteroendocrine cells migrates downward to the bottom of the crypt. Enteroendocrine cells are comprised of subsets, whose differentiation is determined by specific transcription factors that regulate both the specific co-expression of hormones and their location within the crypt-villus axis. The differentiation signaling pathways mediated by Wnt, Hedgehog, Notch, BMP and EphB/ephrin are restricted spatially along the crypt-villus axis in the epithelium and the mesenchyme of the intestine. Below position +4 (where the self-renewing Lgr5+ stem cells reside with Paneth cells) in the crypt, Wnt-signaling is especially active . Presently, it is unclear what determines whether a subset of enteroendocrine cells remains in the Wnt signaling rich area instead of migrating up to the villi and why. Similar to other enteroendocrine cells, EC cells, the presumed cell of origin of the SI-NETs, differentiate from the ISCs. A majority of EC cells are found at higher positions within the crypts and the villi and would eventually be sloughed as they reach the villus tips. However a small proportion of EC cells can be found below +4 position where an active stem cell niche maintains ISCs. Similar to our previous work in the duodenum of mice, we wondered if the EC cells in the distal SI that reside below +4 position were derived from reserve ISCs and were susceptible to tumorigenesis in Familial SI-NET patients. Presently, it is unknown whether there is a subset of ISC marker-expressing enteroendocrine cells in the human small intestine and whether they are related to SI-NETs. Therefore, we are presently investigating the relationship between the expression of ISC marker genes in EC cells along the crypt-villus axis of the human norman ileum and their expressions in SI-NETs. Recent results identified multifocal aberrant crypt-containing endocrine cell clusters (ACECs) that contain crypt EC cell microtumors in patients with familial SI-NETs. RNA in situ hybridization revealed expression of the EC cell and reserve stem cell genes TPH1, BMI1, HOPX, and LGR5(low), in the ACECs and more advanced extraepithelial tumor nests. This expression pattern resembled that of reserve EC cells that express reserve ISC genes; most reside at the +4 position in normal crypts. The presence of multifocal ACECs from separate tumors and in the macroscopic tumor-free mucosa indicated widespread, independent, multifocal tumorigenesis. Analyses of mitochondrial DNA confirmed the independent origin of the ACECs. Our most recent results show that fully differentiated EECs residing predominantly at the +4 position in the crypt have features of both label retaining cells (LRC) and intestinal stem cells (ISC) and are capable of de-differentiating to ISCs to regenerate the epithelium under basal and pathological ISC dynamics. Using lineage tracing in vivo and in ex vivo organoids, we show that the enterochromaffin (EC) cell is the predominant EEC with this potential. These studies suggest that an ISC gene-expressing subset of fully differentiated EC cells contributes to basal and pathological stem cell dynamics in the small intestine. These findings provide novel insights into the +4 reserve ISC hypothesis, stem cell dynamics of the intestinal epithelium and novel insight in the development of EC-derived small intestinal tumors. Take together, our studies suggest that Familial SI-NETs originate from a subset of EC cells (reserve EC cells that express reserve ISC genes) via multifocal and polyclonal processes.